Trisaccharide Derivates, and Their Use as Adjuvants

ABSTRACT

The present invention relates to the use of trisaccharide derivates comprising a substituted trisaccharide core, which trisaccharide core is fully substituted with fatty acid ester groups, and optionally one or more anionic groups as adjuvants, to the trisaccharide derivates as such, to a method for preparing such trisaccharides, to trisaccharides obtained with such method, to adjuvant compositions comprising such trisaccharide derivates and to a vaccine or kit comprising such adjuvant compositions.

FIELD OF THE INVENTION

The present invention relates to novel trisaccharide derivates and theiruse as adjuvants, which trisaccharide derivates comprise a substitutedtrisaccharide core, which trisaccharide core is fully substituted withfatty acid ester groups, and optionally one or more anionic groups, theinvention further relates to a method for preparing these trisaccharidederivates, to trisaccharides obtainable by this method, and to the useof the adjuvant in a vaccine.

BACKGROUND OF THE INVENTION

Antibodies are substances contained in the blood and other fluids of thebody, as well as in the tissues, and which bind to antigen to make itinnocuous. Antibodies constitute one of the natural defense mechanismsof the body. They are highly specific and they kill, bind or makeinnocuous the antigen which induced their formation.

The antigen in contact with the immune system, thus activates a complexseries of cellular interactions to eliminate the antigen and/or tore-establish the preceding equilibrium.

Two of the characteristic features of antigens are their immunogenicity,that is their capacity to induce an immune response in vivo (includingthe formation of specific antibodies), and their antigenicity, that istheir capacity to be selectively recognized by the antibodies whoseorigins are the antigens.

It is known how to stimulate the immune response deliberately byadministering a specific antigen by means of a vaccine. The procedureallows the retention of a state of immune response in the organism whichallows a more rapid and more effective response of the organism duringsubsequent contact with the antigen.

However, some antigens have only a weak immunogenicity and they inducean insufficient immune response to produce an effective protection forthe organism. This immunogenicity can significantly be improved if anantigen is co-administered with an adjuvant.

Adjuvants are substances that enhance the immune response to antigens,but are not necessarily immunogenic themselves. Adjuvants may act byretaining the antigen locally near the site of administration to producea depot effect facilitating a slow, sustained release of antigen tocells of the immune system. Adjuvants can also attract cells of theimmune system to an antigen depot and stimulate such cells to elicitimmune responses.

Adjuvants have been used for many years to improve the host immuneresponse to, for example, vaccines. Intrinsic adjuvants are normally thecomponents of the killed or attenuated bacteria used as vaccines.Extrinsic adjuvants are immunomodulators which are typicallynon-covalently linked to antigens and are formulated to enhance the hostimmune response.

Aluminium hydroxide and aluminium phosphate (collectively referred to asalum) are routinely used as adjuvants in human and veterinary vaccines.The efficacy of alum in increasing antibody responses to diphtheria andtetanus toxoids is well established and, more recently, a HBsAg vaccinehas been adjuvanted with alum.

A wide range of extrinsic adjuvants can provoke immune responses toantigens. These include saponins complexed to membrane protein antigens(immune stimulating complexes), pluronic polymers with mineral oil,killed mycobacteria in mineral oil, Freund's complete adjuvant,bacterial products, such as muramyl dipeptide (MDP).

Chemically defined adjuvants, such as monophosphoryl lipid A,phospholipid conjungates have been investigated (see Goodman-Snitkoff etal., J. Immunol. 147:410-415 (1991) as has encapsulation of the proteinwith a proteoliposome (see Miller et al., J. Exp. Med. 176:1739-1744(1992)).

Synthetic polymers have also been evaluated as adjuvants. These includethe homo- and copolymers of lactic and glycolic acid, which have beenused to produce microspheres that encapsulate antigens (see Eldridge etal., Mol. Immunol. 28:287-294 (1993)).

Nonionic block copolymers are another synthetic adjuvant beingevaluated. Adjuvant effects have also been investigated for lowmolecular weight copolymers in oil-based emulsions (see Hunter et al.,The Theory and Partical Application of Adjuvants (Ed. Stewart-Tull, D.E. S.) John Wiley and Sons, NY. Pp. 51-94 (1995)) and for high molecularweight copolymers in aqueous formulations (Todd et al., Vaccin15:564-570 (1997)).

Desirable characteristics of ideal adjuvants are lack of toxicity and anability to stimulate a long lasting immune response. One of the mostcommonly used adjuvants in humans is alum. Other adjuvants, such asSaponin, Quil A and the water in oil adjuvant, Freund's with killedtubercle bacilli (Freund's complete) or without bacilli (Freud'sincomplete), have had limited use in humans due to their toxic effects;and concerns have been raised as to undesirable effects in animals.

Simply said, many adjuvant formulations have been described but most arenever accepted for routine vaccines, and few have been approved for usein humans. This is mainly due to their toxicity. For example, themineral oils used as adjuvants in certain animal vaccines are notreadily degraded and persist at the site of injection thereby causingunacceptable granulomas; and, in general adjuvant formulations such asmineral compounds oil emulsions, liposomes and biodegradable polymermicrospheres cause local reactions due to depot formation at the site ofinjection.

Examples of adjuvants presently approved in human vaccines include Alum,MF59 (an oil in water emulsion), MPL (a glycolipid), VLR,Immunopotentiating Reconstituted Influenza Virosomes (IRIV) and choleratoxin (see Reed et al. Trends in Immunology 30:23-32 (2008).

One group of adjuvants known in the art are the so calledsulpholipopolysaccharides, i.e. polysaccharides containing both fattyacid esters and sulphate esters (Hilgers et al., Immunology 60, pp.141-146, 1986). A method for preparing these compounds has beendescribed in the international patent application WO96/20222 and WO96/20008. These methods for preparing sulpholipopolysaccharides resultin the formation of different sulpholipopolysaccharides derivatesvarying in the number of fatty acids esters present per polysaccharidemolecule, the number of sulphate esters present per polysaccharidemolecule, the number of hydroxyl groups per polysaccharide molecule andthe distribution of the fatty acid esters, the sulphate esters and thehydroxyl groups over the polysaccharide molecule. This means that duringpreparation of these sulpholipopolysaccharides a mixture is obtained ofmany different sulpholipopolysaccharides. Consequently, the yield of thedesired sulpholipopolysaccharide is relatively low or the adjuvant needsto be used as a difficult to characterise mixture causing regulatoryissues.

In the European patent EP 1233969 an adjuvant composition is claimedwhich adjuvant comprises sulpholipodisaccharides. Also a method isdescribed for preparing these sulpholipodisaccharides. In one of theembodiments the sulpholipodisaccharides prepared are fully substitutedwith fatty acid ester or sulphate ester groups. However, as will befurther described in the following, when these sulpholipodisaccharidesare used as adjuvants in animals, undesired side effects such asoccurrence of mean body temperature rise (including fever) and localirritation (tissue swelling) occur.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: HPLC-ELSD chromatogram of sulpho-lipo-maltotriose,synthesized via pyridine.SO₃ route

FIG. 2: HPLC-ELSD chromatogram of sulpho-lipo-raffinose, synthesized viapyridine.SO₃ route

FIGS. 3A and 3B: HPLC-ELSD chromatogram of lipo-maltotriose andlipo-raffinose

FIGS. 4A, 4B and 4C: GnRH antibody titer (mean) of rats immunized withvarious vaccine formulations

FIGS. 5A, 5B and 5C: Serum testosterone levels of rats immunized withvarious vaccine formulations

FIGS. 6A, 6B and 6C: Mean body temperature of rats immunized withvarious vaccines formulations

FIGS. 7A, 7B and 7C: Injection site reaction of rats immunized withvarious vaccine formulations

DETAILED DESCRIPTION OF THE INVENTION

In view of the foregoing it is an object of the present invention toprovide compounds which are relatively easy and inexpensive to prepare,have good adjuvating properties and induce a minimum of undesired sideeffects when used clinically. It is a further object of the presentinvention to provide compounds that can be used in an adjuvantcomposition, for example in combination with a vaccine, which compoundshave an excellent safety and side effect profile.

A first and second aspect of the present invention relates totrisaccharide derivates and their use as an adjuvant. The trisaccharidederivates according to the present invention comprise a substitutedtrisaccharide core, which trisaccharide core is fully substituted withfatty acid ester groups, and optionally one or more anionic groups.

The trisaccharide derivates according to the present invention arehighly suitable for use as adjuvant for vaccines. They have a sideeffect profile which is surprisingly significantly better than the sideeffect profile of other adjuvants which are based on polysaccharidederivates, such as for example adjuvants based on disaccharides. Animalswhich have been vaccinated with an antigen composition and an adjuvantcomposition comprising the trisaccharide derivates according to theinvention show less increase in mean body temperature compared with forexample the disaccharide derivates of EP 1233969. Also the occurrence oflocal reactions (tissue swelling) around the area of injection is lowerwhen an adjuvant is used which comprises the trisaccharide derivatesaccording the invention.

The term antigen as used herein, refers to any component or materialthat induces a immunological reaction in the human or animal body, suchas a virus, a bacterium, mycoplasma, a parasite or a tumor cell, asubunit of a micro-organism, such as a protein, polysaccharide, peptide,glycoprotein, polysaccharide-protein conjugate,peptide-protein-conjugate.

The antigen can for example consist of or contain one or more liveorganisms, inactivated organisms, or so-called subunits (the latter e.g. prepared synthetically, or by recombinant DNA methods, or isolatedfrom the organisms). The term antigen further refers to any componentthat can induce an immune reaction in the human or animal body.

The trisaccharide core of the derivates of the present invention ispreferably derived from raffinose, melezitose, maltotriose,nigerotriose, maltotriulose or kestose. It is particularly preferredthat the trisaccharide core is derived from raffinose, melezitose ormaltotriose, most preferably raffinose or maltotriose. Thesetrisaccharides have in their normal. i.e. unsubstituted form elevenOH-groups which are available for reactions such as for exampleesterification with fatty acids. However, it is also possible that oneor more, preferably one, of the OH-groups has reacted with an anionicgroup, such that for example a sulphate ester or phosphate ester groupis obtained, preferably a sulphate ester group is obtained.

In a preferred embodiment of the present invention the trisaccharidederivates according to the invention comprises no anionic groups butonly fatty acids groups, preferably identical fatty acid groups.

According to another preferred embodiment the trisaccharide derivatesaccording to the invention comprise one or two anionic groups with tenor nine fatty acid groups, respectively, per substituted trisaccharidecore. Preferably the fatty acid groups are identical.

The term anionic group as used herein refers to a negatively chargedmoiety (i.e. negatively charged at neutral pH or the pH of theenvironment in which the derivate is applied). Such an anionic group mayfor example be a sulphate, a sulphonate or a phosphate. Preferredanionic groups include sulphate ester groups or phosphate ester groups.Examples of such groups are —O—SO₂—ONa or —O—SO₂—ONH₄, —O—SO₂—OTEA (i.e.sulphate triethylammonium).

In a preferred embodiment of the present invention the fatty acid estergroup which is covalently bound to the substituted trisaccharide core isan ester of a straight, branched, saturated or unsaturated fatty acidwith a chain length of 4 to 20 carbon atoms, preferably 6 to 18, morepreferably 8 to 16 carbon atoms, most preferably 10-14 and highlypreferred 12 carbon atoms.

Although it is within the scope of the present invention that thesubstituted trisaccharide core is substituted with more than one type offatty acid ester, it is preferred that only one type is used, i.e. thatall fatty acid esters are identical.

The use of fatty acids is highly preferred, however it is also envisagedby the present invention that other carboxylic acids, preferably closelyrelated to fatty acids, may provide favourable results.

Preferably, the fatty acid ester is the ester of saturated fatty acids,monounsaturated fatty acids or polyunsaturated fatty acids, such asbutyric acid, caproic acid, caprylic acid, capric acid, lauric acid,myristic acid, palmitic acid, palmitoleic acid, stearic acid, ricinoleicacid, vaccenic acid, arachidic acid, gadoleic acid, arachidonic acid,oleic acid or linoleic acid. Most preferably lauric acid.

In a preferred embodiment the substituted trisaccharide core is derivedfrom raffinose, melezitose or maltotriose and is the trisaccharidederivate fully substituted with identical fatty acid ester groups persubstituted trisaccharide, i.e. the trisaccharide core is substitutedwith eleven identical fatty acid ester groups.

In another preferred embodiment the substituted trisaccharide core isderived from raffinose, melezitose or maltotriose and comprises one ortwo anionic groups, such as a sulphate ester or a phosphate ester group,and ten or nine, respectively, identical fatty acid ester groups persubstituted trisaccharide. Most preferably the fatty acid ester is theester of lauric acid.

A third aspect of the present invention relates to a method forpreparing a trisaccharide derivate comprising the steps of:

i) providing a trisaccharide and dissolving it in a solvent; and ii)esterifying all OH-groups of the trisaccharide with a fatty acid, orsource thereof, optionally reacting at least one of the OH-groups of thetrisaccharide with an anionic agent.

Due to the fact that all the OH-groups are reacted with anionic groupsand/or fatty acids, few impurities are being formed. This means that itis possible to obtain without extensive purification steps apharmaceutically acceptable pure form of an envisaged trisaccharidederivate. If only one kind of fatty acid is used, such as lauric acid,even less purification is needed to obtain the desired trisaccharidederivate in a pharmaceutically acceptable pure form. Another advantageof the method of the present invention is that the desired trisaccharidederivates are easily obtained in relatively large quantities, making themethod economically attractive.

In a preferred embodiment of the present invention the trisaccharidederivates prepared comprise no anionic groups but only fatty acidsgroups, preferably identical fatty acid groups, i.e. all OH-groups havereacted with a fatty acid or source thereof.

According to another preferred embodiment the trisaccharide derivatesprepared comprise one or two anionic groups with ten or nine fatty acidgroups, respectively, per substituted trisaccharide core. Preferably thefatty acid groups are identical.

The trisaccharides used in the above mentioned method are preferablyraffinose, melezitose, maltotriose, nigerotriose, maltotriulose orkestose. More preferably, raffinose, melezitose or maltotriose are used,most preferably maltotriose or raffinose.

The meaning of fatty acids as used in the above mentioned method refersto any source of fatty acids, including fatty acid salts, fatty acidshalides, fatty acid esters and derivates. Preferably the fatty acidsused in the claimed method are straight, branched, saturated orunsaturated fatty acids with a chain length of between 4 to 20 carbonatoms, preferably between 6 to 18, more preferably from 8 to 16 carbonatoms, most preferably 10 to 14 carbon atoms, highly preferred 12 carbonatoms.

Preferably, the fatty acids used are saturated fatty acids,monounsaturated fatty acids or polyunsaturated fatty acids, such asbutyric acid, caproic acid, caprylic acid, capric acid, lauric acid,myristic acid, palmitic acid, palmitoleic acid, stearic acid, ricinoleicacid, vaccenic acid, arachidic acid, gadoleic acid, arachidonic acid,oleic acid or linoleic acid. Most preferably lauric acid.

As mentioned above, in a preferred embodiment of the present inventionat least one of the OH groups is reacted with an anionic reagent.Preferably, the anionic agent used is a sulphating or phosphating agentsuch as gaseous SO₃, HCLSO₃, SO₃.pyridine, SO₃-2-methylpyridine,SO₃-2,6-dimethylpyridine, SO₃-dimethylformamide, SO₃-trimethylamide,SO₃-triethylamine, SO₃-dimethylanaline, SO₃—N-ethylmorpholine,SO₃-diethylalanine, SO₃-dioxane and combinations thereof. Mostpreferably the sulphonating agent is SO₃.pyridine or SO₃.triethylamine.

The most preferred sulphating agent is SO₃.pyridine. It is furtherpreferred that reacting at least one of the OH-groups of thetrisaccharide with a sulphating agent is carried out before theesterification of the trisaccharide with a fatty acid. The advantage offirst carrying out the reaction with a sulphating agent is that thesulphating agent reacts first with the so called primary OH-groupsbefore reacting with other OH groups, thereby reducing the number ofisomers formed.

In a preferred embodiment the ratio trisaccharide:anionic agent:fattyacid equivalents is 1:0-3:8-11, preferably 1: 0-1:10-11. Within theseclaimed ranges the complete substitution of the OH-groups of thetrisaccharide with fatty acid esters and optionally the anionic group,such as a sulphate ester, is efficiently obtained.

Preferably, the solvent used for carrying out the reaction is a mixtureof pyridine and dimethylformamide.

In an additional step of the present method the trisaccharide derivatesare subjected to an additional step of mixing them with apharmaceutically acceptable excipient or diluent, such that an adjuvantcomposition is obtained.

A fourth aspect of the present invention relates to adjuvantcompositions comprising trisaccharide derivates according to theinvention, or a mixture thereof. When such trisaccharide derivates areformulated into an adjuvant composition they are preferably mixed withpharmaceutically acceptable excipients or diluents. Preferably theadjuvant composition is formulated as an oil in water emulsion. Suitableoils to be used are amongst others animal oils, vegetable oils andmineral oils, such as fish oil, vitamine E, sqalane, squalene.Preferably, use is made of squalane, preferably in combination withpolysorbate.

Although it is possible to only use one kind of trisaccharide derivateit is also within the scope of the invention to use in the adjuvantcomposition a mixture of different trisaccharide derivates according tothe invention. In a preferred embodiment a mixture is used oftrisaccharide derivates according to the invention with an anionicgroup, such as a sulphate ester group, and the same trisaccharidederivates without an anionic group, e.g. sulpho-lipo-raffinose andlipo-raffinose. Most preferably the fatty acid esters of thetrisaccharides derivates used in such a mixture are the same, such asfor example the ester of lauric acid.

A fifth aspect of the present invention relates to a vaccine comprisingthe adjuvant composition, or trisaccharide derivate as mentioned above.

Both the adjuvant composition, antigen composition or vaccine arepreferably administered parenterally. Suitable means for parenteraladministration include intramuscular, subcutaneous, subdermal andintradermal administration. Suitable devices for parenteraladministration include needle (including microneedle) injectors, andtransdermal delivery systems.

The parenteral formulation may readily be prepared by someone skilled inthe art according to standard methods. Preferably the parenteralformulation is prepared as an oil in water emulsion.

The preparation of parenteral formulations under sterile conditions, forexample, by filtration may readily be accomplished using standardpharmaceutical techniques well known to those skilled in the art.

The vaccine or the adjuvant composition according to the invention canbe administered to humans and many different target animals, such as forexample pigs, cattle, poultry, dogs, cats, horses and the like.

A sixth aspect of the present invention relates to a kit comprising theabove mentioned adjuvant composition and an antigen composition.

It may be desirable to administer a combination of the adjuvantcomposition and antigen composition or vaccine separately. In such acase the adjuvant composition and antigen composition may convenientlybe combined in the form of a kit. Such a kit could for example be a twovial system or a dual chamber syringe.

The invention will now be further described by the following,non-limiting examples

EXAMPLES Preparation of Trisaccharide Derivates According to theInvention Example 1 General Synthesis of Sulpho-Lipo-TrisaccharidesAccording to the Invention

A trisaccharide is dried in a vacuum oven to remove the crystal water.The trisaccharide (5 g) is subsequently dissolved, under a stream ofnitrogen, in 30 mL of DMF and 14 mL of pyridine in a 100 mL three-neckround-bottom flask, equipped with a reflux-condensor. 1.05 equivalent ofpyridine.SO₃ is added under vigorous stirring. After 1 hr the flask iscooled in ice water and under vigorous stirring lauroyl chloride isadded drop-wise to prevent heating of the reaction mixture. After 15 minthe ice bath is slowly heated to 40° C. The progress of the reaction ismonitored by HPLC. After completion of the reaction the mixture isconcentrated in vacuo in a rotary evaporator (heated up to 60° C.). Thecrude product is taken up in 300 mL heptane and 150 mL brine. Theorganic layer is separated in a 500 mL separatory funnel, dried onsodium sulfate and filtered. The heptane solution is concentrated invacuo. The oil obtained is dissolved in 200 mL heptane and triethylamine(2.37 mL) is added drop wise. The solution obtained is filtered andconcentrated in vacuo. The oil is dissolved again in 100 mL heptane,filtered and the solution is concentrated in vacuo.

Example 2 Synthesis of Sulpho-Lipo-Maltotriose (S1L10-Ma) ViaPyridine.SO₃ Route

The title product was synthesized following the general method describedin Example 1 with the following details: maltotriose (5.1 g, 10 mmol)was dried in a vacuum stove at <10 mbar for 19 hrs at 40° C. and 48 hrsat 70° C., yield 4.9 g. The dried maltotriose was dissolved in 30 mL ofDMF and 14 mL of pyridine and sulfated with 1.6 g (1.05 eq) ofpyridine.SO₃, suspended in 2 mL of DMF. After 1 hr thesulfo-trisaccharide was esterified in an ice bath with 11 equivalents(25.5 mL) of lauroyl chloride. The mixture was slowly heated to 40° C.and reacted for 3 hrs. Reaction steps were followed by HPLC-ELSD. Theproduct was isolated after extraction and triethylamine exchange. Yieldof the thick brown-yellow syrup: 15.5 g+5.5 g (second crop fromevaporation flask after heating to 50° C.). HPLC-ELSD chromatogram: seeFIG. 1A.

Example 3 Alternative Synthesis of Sulpho-Lipo-Maltotriose (S1L10-Ma)Via Pyridine.SO₃ Route

The title product was synthesized following the general method describedin Example 1 with the following details: maltotriose (5.0 g, 10 mmol)was dried in a vacuum stove at <10 mbar for 20 hrs at 40° C. and 90 hrsat 70° C., yield 4.9 g. The dried maltotriose was dissolved in 30 mL ofDMF and 1.6 g (1.05 eq) of pyridine.SO₃ was added as a suspension in 14mL of pyridine. After 1 hr the sulpho-trisaccharide was esterified in anice bath with 11 equivalents (25.2 mL) of lauroyl chloride and wasslowly heated to 40° C. Samples for HPLC-ELSD analysis were taken atregular time points in the process. The reaction with lauroyl chloridewas left overnight at ambient temperature Work up was done as describedin example 1. Yield of the thick brown-yellow syrup: 24.7 g. HPLC-ELSDchromatogram: see FIG. 1B.

Example 4 Synthesis of Sulpho-Lipo-Raffinose (S1L10-Ra) Via Pyridine.SO₃Route

The title product was synthesized as described in example 1 with thefollowing details: raffinose pentahydrate (5.0 g, 10 mmol) was dried ina vacuum stove at <10 mbar for 24 hrs at 30° C. and 90 hrs at 60° C.,yield 4.3 g. The dried raffinose was dissolved in 30 mL of DMF and 14 mLof pyridine. Pyridine.SO₃ (1.6 g, 1.05 eq) was added in one batch. After1 hr the sulpho-trisaccharide was esterified in an ice bath with 12equivalents (23.5 mL) of lauroyl chloride and was slowly heated to 40°C. After 4 hrs, the mixture was concentrated in vacuo, extracted andtreated with TEA as described in example 1. Yield of the thickyellow-brown syrup: 18.0 g. HPLC-ELSD chromatogram: see FIG. 2.

Example 5 Synthesis of Lipo-Maltotriose (L11-Ma)

The title product was synthesized following the general method describedin Example 1 with the following details: Maltotriose hydrate (0.50 g)was dried in a vacuum oven. The dried trisaccharide (0.49 g, 0.97 mmol)was dissolved in pyridine (1.4 mL) and DMF (3 mL), cooled in an ice bathand reacted with lauroyl chloride (3.36 mL, 15 eq) for 1 hrs at 0° C.,followed by 16 hrs at room temperature. The reaction mixture became agel, upon the addition of 3 mL of heptanes and sonification the productdissolved. The addition of heptane was repeated twice. The organic phase(75 mL) was washed with water and a three layer system was formed. Onlythe organic layer was isolated, dried over sodium sulphate, filtered andconcentrated to yield a thick yellow-brown syrup: 1.39 g. HPLC-ELSDchromatogram: see FIG. 3A.

Example 6 Synthesis of Lipo-Raffinose (L11-Ra)

Raffinose pentahydrate (0.50 g) was dried in a vacuum oven. The driedraffinose (0.41 g, 0.86 mmol) in pyridine (1.4 mL) and DMF (3 mL) wascooled in an ice bath and reacted with lauroyl chloride (2.97 ml, 15 eq)for 1 hrs at 0° C., followed by 18 hrs at room temperature. The reactionmixture was diluted with heptane (50 ml) and washed with water (25 ml).The organic phase was dried over sodium sulphate, filtered andconcentrated to yield a thick yellow-brown syrup: 2.33 g. HPLC-ELSDchromatogram: see FIG. 3B.

Effect of Adjuvants Comprising the Trisaccharide Derivates According tothe Invention on Anti GnRH Titers, Serum Testosterone Levels andOccurrence of Adverse Effects.

Animal experiments have been carried out to assess the efficacy andpossible adverse effects associated with the use of the trisaccharidesaccording to the invention as an adjuvant. In this study in rats, three(sulpho-) lipo-trisaccharides based adjuvants were tested:sulpho-lipo-maltotriose (one sulphate-ester group and ten lauroyl estergroups, S1L10-Ma), prepared according to Example 3;sulpho-lipo-raffinose (one sulphate-ester group and ten lauroyl estergroups, S1L10-Ra), prepared according to Example 4; and lipo-raffinose(raffinose fully substituted with lauroyl ester groups, L11-Ra),prepared according to Example 6. The adjuvants comprisingsulpho-lipo-maltotriose also comprised lipo-maltotriose (fullysubstituted with lauroyl ester groups, L11-Ma). The adjuvants comprisingsulpho-lipo-raffinose also comprised lipo-raffinose (fully substitutedwith lauroyl ester groups, L11-Ra).

The sulpho-lipo based adjuvants were tested in different doses varyingfrom 0.008-8 mg per dose. The adjuvants were tested in combination witha GnRH-KLH conjugate with 0.7 μg conjugated GnRH per dose. Adjuvanticityof the adjuvants was compared with the positive control adjuvant,sulpho-lipo-sucrose (disaccharide with one sulphate ester group andseven lauroyl acid ester groups, S1L7-Su) and with negative controladjuvant consisting of an squalane-in-water emulsion without saccharidecompound and one group receiving PBS only.

Efficacy was determined by antibody titers and biological effects of theinduced antibodies on testosterone levels. In order to determine adverseeffects of immunization with the adjuvants, daily clinical observationswere made and body temperature and injection site reactions wasdetermined.

Preparation of Adjuvant Emulsions

-   -   Saccharides: Sulpho-lipo-maltotriose (S1L10-Ma)        -   Sulpho-lipo-raffinose (S1L10-Ra)        -   Lipo-raffinose (L11-Ra)    -   Squalane (A&E Connock)    -   Polysorbate-80 (Fagron)    -   Sterile PBS-wit pH 7.4 (Mediabereiding ASG, Lelystad)    -   MilliQ water    -   Millex syringe driven filter unit 0.22 μm PES, 33 mm, 4.5 cm²        (Millipore)    -   Microfluidizer M-110S equipped with interaction chamber type:        F20Y    -   Microtrac Nanotrac Analyzer System NPA-253

The experimental adjuvants were prepared as follows.

TABLE 1 Composition of the emulsions for the different SLS AmountSqualane Tween- MilliQ PBS- Group Saccharide SLS (g) (g) 80 (g) (g) wit(g) 1 Sulpho-lipo-maltotriose 1.0018 4.0010 1.0019 0.2507 18.7495(S1L10-Ma-8) 2 Sulpho-lipo-maltotriose 0.2497 4.0051 1.0017 1.000718.7515 (S1L10-Ma-2) 3 Sulpho-lipo-maltotriose 0.1007 4.0025 1.00301.1525 18.7524 (S1L10-Ma-0.8) 4 Sulpho-lipo-maltotriose 0.0101 4.00401.0010 1.2439 18.7525 (S1L10-Ma-0.08) 5 Sulpho-lipo-maltotriose 0.002259.0018 1.0006 1.2706 18.75 (S1L10-Ma-0.008) 6 Sulpho-lipo-raffinose1.0029 4.0004 1.0009 0.2528 18.7495 (S1L10-Ra-8) 7 Sulpho-lipo-raffinose0.2503 4.0007 1.0008 1.0015 18.7514 (S1L10-Ra-2) 8 Sulpho-lipo-raffinose0.1004 4.0015 1.0020 1.1496 18.7526 (S1L10-Ra-0.8) 9Sulpho-lipo-raffinose 0.0106 4.0003 1.0008 1.2386 18.7529(S1L10-Ra-0.08) 10 Sulpho-lipo-raffinose 0.00154 6.1587 1.0045 1.252618.76 (S1L10-Ra-0.008) 11 None 0.0000 3.9995 1.0011 1.2527 18.74 12Lipo-raffinose (L11-Ra-8) 1.0006 4.0067 1.0037 0.2502 18.76 Note: forgroup 5 and 10 the saccharide was dissolved in squalane. Of thesesolutions 4.0056 g (5) and 4.0006 g (10) was used for further emulsionpreparation. The SLS content in the final emulsion was 1 mg/25 ml.

The oil phase components (Saccharide, squalane, polysorbate-80 andMilliQ water, amounts as indicated in table 1) were weighed in a 50 mlFalcon tube. The components were mixed using a vortex and heated in awater bath at 50° C. until the saccharide was dissolved. The warm oilphase was added to the water phase (PBS) and the two phases were mixedby vortex and ultra-turrax at 24000 min⁻¹ for approximately 30 sec withintervals. Subsequently, the emulsion was formed by Microfluidizerprocessing. The operating pressure was set to 500 kPa (5 bar) and eachmixture was passed three times under cooling of the interaction chamberin an ice bath. Each emulsion was subjected to manual sterilefiltration. The particle size of the emulsion was measured using aNanotrac particle sizer.

Vaccines 1-13 were formulated by adding equal volume of adjuvant(comprising the saccharide compounds as shown in table 2) to the waterphase (containing 0.7 μg conjugated GnRH). Vaccine 14 consists of PBSonly. The following vaccines were prepared:

TABLE 2 Experimental design Group Saccharide compound Adjuvant dose 1sulpho-lipo-maltotriose (S1L10-Ma-8) 8 mg 2 sulpho-lipo-maltotriose(S1L10-Ma-2) 2 mg 3 sulpho-lipo-maltotriose (S1L10-Ma-0.8) 0.8 mg 4sulpho-lipo-maltotriose (S1L10-Ma-0.08) 0.08 mg 5sulpho-lipo-maltotriose (S1L10-Ma-0.008) 0.008 mg 6sulpho-lipo-raffinose (S1L10-Ra-8) 8 mg 7 sulpho-lipo-raffinose(S1L10-Ra-2) 2 mg 8 sulpho-lipo-raffinose (S1L10-Ra-0.8) 0.8 mg 9sulpho-lipo-raffinose (S1L10-Ra-0.08) 0.08 mg 10 sulpho-lipo-raffinose(S1L10-Ra-0.008) 0.008 mg 11 no saccharide (NS) 0 12 lipo-raffinose(L11-Ra) 8 mg 13 sulpho-lipo-sucrose (S1L7-Su-8) 8 mg 14 PBS control (noantigen, no adjuvant) 0

Animals and Immunization

Male Wistar rats, 10 weeks of age, were housed with 3 rats per cage.Rats had ad libitum access to food en water. Rats were immunized at day0, 14 and 28, according to the experimental design (Table 2) with 400 μlvaccine comprising 200 μl water phase and 200 μl adjuvant. Twointramuscular injections (100 μl each) were injected at the left andright inner thigh and 2×100 μl was injected subcutaneously in the neckregion. Each group consists of 5 rats.

Blood samples for serum were collected from all animals prior to theimmunizations and at day 41 and 56

Efficacy of the Vaccines Assays

GnRH specific antibodies were measured by ELISA. Plates (96 wells) wereprecoated with 0.2% glutardialdehyde in phosphate buffer (pH 5) for 3hours at room temperature, washed with 0.1 M phosphate buffer (pH 8) andcoated with 100 μl per well of a solution containing 10 μg GnRH (PepscanPresto, Lelystad, The Netherlands) per ml phosphate buffer (pH 8) andincubated for 3 hours at 37° C. Coated plates were washed with 0.05%Tween-80. Serum samples were diluted ( 1/10) in PEM (1% Tween-80 with 4%horse serum). This dilution was further diluted in the 96-well plate(100 μl per well, 8 steps) and incubated 1 hour at 37° C. After washingwith 0.05% Tween-80, 100 μl of goat-anti-rat antiserum conjugated withperoxidase in PEM was added to the wells. Plates were incubated for 1 hat 37° C. and washed 12 times with 0.05% Tween-80. Subsequently, 150 μlof a substrate solution containing2,2-azino-bis-(3-benzthiazoline-6-sulphonic acid) (ABTS) plus H₂O₂ wasadded to the wells of the plates. Plates were incubated for 45 minutesat ambient room temperature and absorbance was measured at 405 nm.Antibody titer was expressed as the 10 log of the dilution factor thatgives an optical density of 4 times background (approx. 100).

Serum testosterone levels were measured using a commercially availableTestosterone EIA (Beckman Coulter, Woerden, The Netherlands) accordingto the instructions of the manufacturer.

GnRH Antibody Titers

GnRH antibody titers of groups treated with sulpho-lipo-maltotriose(S1L10-Ma) and sulpho-lipo-raffinose (S1L10-Ra) are depicted in FIGS. 4Aand 4B. Both sulpho-lipo-trisaccharides induced dose dependent antibodytiters against GnRH. Antibody titers were substantially higher for ratstreated with 0.08-8 mg sulpho-lipo-trisaccharide than in rats treatedwith adjuvant without saccharide (NS, group 11), demonstrating thesignificant contribution of the sulpho-lipo-trisaccharide to the immuneresponse.

FIG. 4C clearly shows that GnRH antibody titers of rats that received 8mg saccharide compounds according to the invention, i.e. S1L10-Ma-8,S1L10-Ra-8 and L11-Ra-8 were substantially higher than in rats treatedwith squalane emulsion only (NS), emphasizing strong adjuvanticity ofboth S1L10- and L11-trisaccharides.

Serum Testosterone

Immunization with GnRH-KLH conjugate emulsified with the adjuvantcomprising the sulpho-lipo-trisaccharides according to the invention,i.e. S1L10-Ma and S1L10-Ra, resulted in a dramatic decline of serumtestosterone levels from 0.08 mg sulpho-lipo-trisaccharide onwards (FIG.5 A-B), while the effects of the lowest sulpho-lipo-trisaccharide dose(0.008 mg) on serum testosterone were similar to the oil-in-wateremulsion without saccharide compound Immunization with the lipidatedtrisaccharide (L11-Ra-8) also induced decreasing testosterone levels(FIG. 5C).

Adverse Effects Rise of Mean Body Temperature

At least once daily clinical observations were made in all animals. Meanbody temperature (MBT) was determined for all animals before and aftereach immunization by rectal temperature. Mean body temperature per group(MBT) are depicted as to preimmunization levels FIG. 6.

At 3 hours after each vaccination, a slight increase in MBT was observedin rats treated with sulpho-lipo-trisaccharides only at 8 and 2 mg dose,while no effects on MBT were noted at 0.8, 0.08 and 0.008 mg. The nextday 21 hours after vaccination however, MBT was dropped to almost normalvalues again (FIGS. 6A and 6B). In contrast, immunization with thedisaccharide compound (S1L7-Su-8) which caused a slightly higherincrease in MBT than the trisaccharide compounds at 3 hours postvaccination, did not show a decrease in MBT one day post immunization,MBT was still increased at 21 hours after vaccination (FIG. 6C). Thelipidated trisaccharide (L11-Ra-8) did not induce any increase in MBT.From FIG. 6 it is thus clear that the vaccines comprising the compoundsaccording to the invention (S1L10-trisaccharides) as adjuvants inducedsurprisingly significantly shorter temperature effects when compared tothe disaccharide derivates known in the prior art, while the lipidatedtrisaccharide (L11-trisaccharide) in similarity to the oil-in-wateremulsion without a saccharide compound (NS) did not induce anytemperature effects at all.

Injection Site Reactions

Before vaccination the injection sites were assessed for the presence ofabnormalities or existing local reactions. If such abnormalities orlocal reactions were absent, the animal was injected on that site. Aftereach immunization, injection sites were inspected for tissue swelling.Size of subcutaneous injection sites was measured (diameter in mm).Since the intramuscular injection site reactions in the hind leg isdifficult to determine, only the presence of intramuscular injectionssites (tissue swelling) was determined and expressed in arbitrary values(present=10 mm, not present=0 mm). Scores of injection site reactionswere added up per rat for the four injection sites at each inspection,means per group were calculated The results thereof are presented inFIG. 7.

After immunization, minor dose dependent injection site reactions wereobserved in rats treated with sulpho-lipo-trisaccharides, mainly in ratstreated with 8 and/or 2 mg (FIGS. 7A and 7B). Size of the injection sitereactions gradually decreased at day 3 after immunization and werealmost undetectable at day 5 after the subsequent immunization.Injection site reactions caused by immunization withsulpho-lipo-disaccharide (S1L7-Su-8) showed a completely differentpattern: Injections site reactions increased in size up to day 4 andwere more than 4 times bigger than sulpho-lipo-trisaccharides (see FIG.7C), moreover at the final inspection 5 days after immunization stillsignificant injection site reactions were present.

The lipidated trisaccharide (L11-Ra-8) did not induce any adverseeffects at the site of injection. Clearly, sulpho-lipo-disaccharidecomprising vaccine formulations induced more injection site reactionsthan vaccines comprising sulpho-lipo-trisaccharides according to theinvention.

1-10. (canceled)
 11. A trisaccharide derivate comprising a substitutedtrisaccharide core, which trisaccharide core is fully substituted withfatty acid ester groups, and optionally one or more anionic groups as anadjuvant.
 12. The trisaccharide derivate according to claim 11, whereinthe substituted trisaccharide core is derived from raffinose,melezitose, maltotriose, nigerotriose, maltotriulose or kestose.
 13. Thetrisaccharide derivate according to claim 11, wherein the substitutedtrisaccharide core comprises one or two sulphate ester or phosphateester groups as anionic groups.
 14. The trisaccharide derivate accordingto claim 11, wherein the anionic group is a sulphate ester.
 15. Thetrisaccharide derivate according to claim 11, wherein the fatty acidester group is an ester of a straight, branched, saturated orunsaturated fatty acid with a chain length of 4 to 20 carbon atoms. 16.The trisaccharide derivate according to claim 11, wherein the fatty acidester is the ester of lauric acid, myristic acid, palmitic acid, stearicacid or arachidic acid.
 17. The trisaccharide derivate according toclaim 11, wherein the fatty acid ester groups of the substitutedtrisaccharide core are all identical.
 18. The trisaccharide derivateaccording to claim 11, wherein the substituted trisaccharide core isderived from raffinose, melezitose or maltotriose and wherein thetrisaccharide derivate is fully substituted with identical fatty acidester groups per substituted trisaccharide.
 19. The trisaccharidederivate according to claim 11, wherein the substituted trisaccharidecore is derived from raffinose, melezitose or maltotriose and whereinthe trisaccharide core comprises one sulphate ester or phosphate estergroup and ten identical fatty acid ester groups per substitutedtrisaccharide or two sulphate or phosphate ester groups and nineidentical fatty acid ester groups per substituted trisaccharide.
 20. Thetrisaccharide derivate according to claim 11, wherein the fatty acidester groups are the esters of lauric acid.
 21. A method for preparing atrisaccharide derivate comprising the steps of: i) providing atrisaccharide and dissolving it in a solvent; and ii) esterifying allOH-groups of the trisaccharide with a fatty acid, or source thereof. 22.The method according to claim 21, wherein at least one of the OH-groupsof the trisaccharide is reacted with an anionic agent.
 23. The methodaccording to claim 22, wherein the anionic agent is a sulphating agentsuch as pyridine.SO₃ or TEA.SO₃ or a phosphating agent.
 24. The methodaccording to claim 21, wherein the solvent used is dimethylformamide,pyridine or a mixture thereof.
 25. The method according to claim 21,wherein the ratio trisaccharide:anionic agent:fatty acid equivalents is1:0-3:8-11 respectively.
 26. The method according to claim 21, whereinthe trisaccharide is raffinose, melezitose, maltotriose, nigerotriose,maltotriulose or kestose.
 27. The method according to claim 21, whereinthe trisaccharide is raffinose, melezitose or maltotriose.
 28. Themethod according to claim 21, wherein the fatty acid is a straight,branched, saturated or unsaturated fatty acid with a chain length ofbetween 4 to 20 carbon atoms.
 29. The method according to claim 21,wherein the fatty acid is lauric acid, myristic acid, palmitic acid,stearic acid or arachidic acid.
 30. The method according to claim 21,wherein the trisaccharide is raffinose, melezitose or maltotriose andwherein the trisaccharide is fully esterified with identical fatty acidsor with on average one sulphate ester or phosphate ester group and tenidentical fatty acid ester groups per trisaccharide or two sulphate orphosphate ester groups and nine identical fatty acid ester groups pertrisaccharide. 31-33. (canceled)
 34. An adjuvant composition comprisinga trisaccharide derivate according to claim 11 and a pharmaceuticalacceptable recipient and/or diluent.
 35. The adjuvant compositionaccording to claim 34 formulated as an oil in water emulsion.
 36. Theadjuvant composition according to claim 35 wherein the oil phase of theemulsion comprises squalane and/or polysorbate. 37-38. (canceled)